| HS Code | 377588 |
| Product Name | Fluorenyl Moxycarbonyl Succinimide (Fmoc-OSu) |
| Synonyms | N-(9-Fluorenylmethoxycarbonyloxy)succinimide, Fmoc-NHS |
| Iupac Name | (9H-fluoren-9-yl)methyl 2,5-dioxopyrrolidin-1-yl carbonate |
| Cas Number | 82911-69-1 |
| Molecular Formula | C19H15NO5 |
| Molecular Weight | 337.33 g/mol |
| Appearance | White to off-white crystalline solid |
| Melting Point | 148-150 °C |
| Purity | ≥98% |
| Solubility | Soluble in DMF, dichloromethane, acetonitrile, THF; sparingly soluble in water |
| Storage Conditions | Store at 2-8 °C, protected from moisture, light, and in a desiccator |
| Applications | Reagent for introducing Fmoc protecting group to amines in peptide synthesis |
| Reactivity | Reacts with primary and secondary amines to form stable Fmoc-carbamates |
As an accredited Fluorenyl Moxycarbonyl Succinimide factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Supplied as a white crystalline powder in a sealed glass bottle, typically available in 5 g or 25 g quantities. |
| Container Loading (20′ FCL) | 20' FCL: pack Fluorenyl Moxycarbonyl Succinimide in dry, sealed drums; secure load, label properly, and ensure ventilation to prevent moisture damage. |
| Shipping | Fluorenyl Moxycarbonyl Succinimide (Fmoc-OSu) should ship in sealed, moisture-resistant containers under inert atmosphere, protected from light and heat. Use desiccants and temperature-controlled packaging to prevent hydrolysis. Ensure compliance with hazardous material regulations and clearly label for handling. Avoid prolonged transit delays to maintain reactivity and purity. |
| Storage | Store Fluorenyl Moxycarbonyl Succinimide (Fmoc-OSu) at –20°C in a tightly sealed, desiccated container, protected from light and moisture. Allow the vial to reach room temperature before opening to prevent condensation. Minimize repeated freeze-thaw cycles. Under these conditions, the reagent remains stable for long-term use. |
| Shelf Life | Shelf life is typically 2 years when stored dry at -20°C, protected from moisture and light. |
Fluorenyl methoxycarbonyl succinimide (Fmoc-OSu, CAS 82911-69-1, molar mass 337.33 g/mol) is consumed at the largest downstream volume in the manufacture of Fmoc-protected L-amino acids that feed solid-phase peptide synthesis. The reagent is not applied directly to resin-bound chains in most production workflows; instead, its carbamate-forming reaction with free amino acids is executed in aqueous-organic media before the Fmoc-amino acid enters the synthesizer. The reaction is run with the amino acid dissolved in water–dioxane or water–THF at a volume ratio of 1:1 to 3:2 and maintained at 0–25 °C depending on the substrate. Sodium carbonate is charged at 1.0–1.5 molar equivalents relative to the amino acid to keep the amino group deprotonated while limiting Fmoc-OSu hydrolysis. Fmoc-OSu is typically added at 1.05–1.3 molar equivalents as a solution in dioxane or THF, with the addition rate controlled by a jacketed glass-lined reactor and recirculating chiller to avoid exothermic excursions above 25 °C. The pH is held between 8.5 and 10.5; below 8.5 the free amino group protonates and acylation slows, while above 10.5 the Fmoc group undergoes progressive β-elimination to dibenzofulvene, producing yellow discoloration and oligomeric impurities. After stirring for 6–18 h, the batch is quenched by slow acidification with 2 mol/L hydrochloric acid to pH 2.0–2.5. The product is extracted into ethyl acetate, washed with water and brine, and crystallized from ethyl acetate/n-heptane. Liberated N-hydroxysuccinimide partitions into the aqueous phase, while residual Fmoc-OSu is destroyed in the aqueous quench. Vacuum drying at 40–50 °C to moisture ≤ 0.5% w/w yields material suitable for peptide synthesis. Lot release includes reverse-phase HPLC at 254 nm with area percentage ≥ 99.0%, chiral HPLC for D-enantiomer ≤ 0.2%, and Karl Fischer water determination following USP <921>.
| Amino acid substrate | Solvent system | Fmoc-OSu molar equivalents | Temperature | Post-reaction purity |
|---|---|---|---|---|
| Glycine | water/dioxane 1:1 v/v | 1.05 | 0–5 °C | ≥ 99.0% HPLC |
| L-Phenylalanine | water/THF 3:2 v/v | 1.1 | 20–25 °C | ≥ 98.5% HPLC |
| H-Lys(Boc)-OH | water/dioxane 1:1 v/v | 1.2 | 10–15 °C | ≥ 98.0% HPLC, epimer ≤ 0.2% |
| H-Arg(Pbf)-OH | water/dioxane/0.2 mol/L NaHCO3 | 1.15 | 20–25 °C | ≥ 98.5% HPLC |
Side-chain-protected substrates such as Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Cys(Trt)-OH require the corresponding ω-amino-protected amino acids because those side-chain protecting groups remain orthogonal to the Fmoc-OSu reaction. For these derivatives, industrial batches run at the lower end of the moisture window and use slower Fmoc-OSu addition to avoid partial dissolution of the starting amino acid and subsequent acylation of the carboxylate to form Fmoc dipeptide. The resulting Fmoc-amino acids are then dried to a fine free-flowing powder and filled into double-lined HDPE containers under nitrogen. Batch-to-batch variance is commonly controlled by HPLC area response, loss on drying, and free amine by TLC/chloranil. The most frequent production bottleneck observed in kilogram-scale campaigns is not the primary acylation but rather emulsion formation during ethyl acetate extraction when residual dioxane content is high; the mitigation is partial concentration under reduced pressure before acidification.
In solution-phase peptide programs, Fmoc-OSu is used to cap the N-terminus of unprotected peptide fragments so that the fragment can be stored, purified, and later coupled without amine-side reactions. The protection is run in anhydrous dichloromethane or DMF under nitrogen, using 1.1–1.4 molar equivalents of Fmoc-OSu relative to free amino groups and 1.2–2.0 molar equivalents of N-methylmorpholine or diisopropylethylamine. The mixture is held at 0–10 °C for the first 2 h and then warmed to 20–25 °C for an additional 4–12 h. Completion is confirmed by ninhydrin or chloranil staining on TLC; residual free amine is quenched with acetic anhydride if incomplete, but overacylation must be avoided because Fmoc-OSu can react slowly with hydroxyl groups on serine, threonine, or tyrosine side chains at extended reaction times. The isolated product is washed with 0.5 mol/L citric acid and 5% sodium bicarbonate, dried over anhydrous magnesium sulfate, and triturated with cold methyl tert-butyl ether. Fragments protected by this route are compatible with tert-butyl ester, Boc, trityl, and Pbf side-chain protecting groups, but incompatible with piperidine, DBU, or primary alkylamines in subsequent downstream steps. Typical isolated yields for fragment N-Fmoc protection range from 75% to 92%, with racemization determined by Marfey’s reagent HPLC using L- and D- amino acid standards. The process is particularly useful for medium-length peptide sequences of 30–50 residues that are assembled in protected fragments to avoid the deletion and truncation complexity of linear SPPS. After fragment condensation, the Fmoc group is removed with 20% piperidine in DMF or with 2% DBU in DMF, but the latter requires rigorous exclusion of moisture and a scavenger for liberated dibenzofulvene. The reaction mixture is then quenched with aqueous citric acid and purified by normal-phase silica or reverse-phase C18 preparative HPLC. Published data for exact epimerization rates in sterically hindered fragments is limited; therefore, process development studies should compare the Pbf/t-Bu-protected fragment versus the corresponding unprotected side-chain variant before scale-up.
The Fmoc-OSu-derived amino acid enters automated solid-phase peptide synthesis in resin substitution ranges of 0.3–0.8 mmol/g. Deprotection is performed with 20% piperidine in DMF at 20–25 °C, typically in two stages of 5 min each. Microwave synthesizers can shorten the same operation to 2 min at 75 °C but raise the risk of aspartimide formation at Asp-Gly and Asp-Ser motifs when the cumulative temperature exposure exceeds 70 °C for more than 10 min. Coupling uses 3–5 molar equivalents of Fmoc-amino acid, 3–5 equivalents HATU or HBTU, and 6–10 equivalents N,N-diisopropylethylamine in DMF or NMP, with recirculation pumps maintaining resin slurry homogeneity in batch reactors of 10–500 L working volume. DIC/Oxyma Pure systems are selected for sequences prone to lactam formation because they generate less nitrilium salt-related breakdown; however, Oxyma Pure requires anhydrous DMF with water content ≤ 0.1% w/w. UV monitoring at 301 nm follows the dibenzofulvene–piperidine adduct and provides real-time deprotection yield; a decrease in the integrated peak area signals incomplete Fmoc removal from aggregated β-sheet-prone sequences. Process bottlenecks in kilogram-scale peptide campaigns include resin batch-to-batch swelling variation in DMF, inefficient deprotection in the interior of crosslinked polystyrene resins above 0.6 mmol/g loading, and piperidine waste neutralization before discharge to pH 6–8. The equipment used includes jacketed glass or Hastelloy C-22 reactors, nitrogen-purged pressure filters, and reverse-phase C18 preparative columns of 150–300 mm diameter with mobile phases containing 0.1% trifluoroacetic acid. The Fmoc cleavage window is most sensitive at the N-terminal residues of polyalanine or polyvaline stretches, where incomplete deprotection produces deletion peptides that co-elute with the target peptide on analytical HPLC. In such cases, the standard deprotection time is extended to 2 × 10 min or the piperidine concentration is raised to 30% in DMF, with UV monitoring used to confirm dibenzofulvene release before proceeding to coupling.
In peptide API contract manufacturing organizations, Fmoc-OSu lot qualification includes identity by FTIR against a reference spectrum, assay by HPLC-UV at 254 nm, residual N-hydroxysuccinimide ≤ 0.5% w/w, and water ≤ 0.1% w/w by Karl Fischer titration. Residual solvents in Fmoc-OSu and in derived Fmoc-amino acids are tested against ICH Q3C limits: acetonitrile ≤ 410 ppm, dichloromethane ≤ 600 ppm, and DMF ≤ 880 ppm when those solvents are used in manufacturing. For peptide APIs, Fmoc-derived process impurities include dibenzofulvene, the piperidine adduct of dibenzofulvene, Fmoc-protected late-eluting deletion sequences, and residual N-hydroxysuccinimide. These are separated by C18 reverse-phase HPLC using 0.1% TFA/acetonitrile gradients and quantified by LCMS with electrospray ionization. Release and stability testing follow ICH Q6A for specification setting and ICH Q7 for GMP operations. Fmoc-OSu must be stored in a desiccated container at 2–8 °C and dispensed under nitrogen or argon; repeated ambient exposure above 30 °C or relative humidity above 60% causes hydrolysis to Fmoc-OH and N-hydroxysuccinimide, followed by dibenzofulvene formation and yellowing. Batch records indicate that residual HOSu levels can vary from 0.05% to 0.4% w/w depending on supplier drying efficiency, which affects the exact molar charge in subsequent reactions. The control matrix below is used at raw material release and in peptide API process development.
| Control target | Analytical method | Typical limit |
|---|---|---|
| Fmoc-OSu assay | HPLC-UV 254 nm | ≥ 99.0% area |
| Residual N-hydroxysuccinimide | HPLC-UV | ≤ 0.5% w/w |
| Water | Karl Fischer coulometry USP <921> | ≤ 0.1% w/w |
| Residual solvents | GC-headspace USP <467> | ICH Q3C class limits |
| Peptide API total Fmoc-related impurities | LCMS ESI | ≤ 0.5% total |
The table is not exhaustive for every peptide program; additional tests for elemental impurities by USP <233> ICP-MS and for microbial contamination by USP <61> are applied when the peptide is intended for parenteral administration. Operational boundaries are defined by the moisture sensitivity of Fmoc-OSu and by the base-labile nature of the Fmoc group. Contact with concentrated ammonia, hydrazine, or strongly nucleophilic solvents at ambient temperature leads to premature deprotection and should be avoided. Incompatibility with primary and secondary alkylamines extends to common laboratory buffers such as Tris, glycine, and ethanolamine, which must not be used as quenching agents if the Fmoc group is to remain intact.
Cosmetic peptide manufacturers that produce acetyl hexapeptide-8, palmitoyl pentapeptide-4, and copper tripeptide-1 use Fmoc-OSu-derived Fmoc amino acids on automated solid-phase synthesizers, after which the peptide is cleaved from the resin with TFA/triisopropylsilane/water at 95:2.5:2.5 v/v/v for 2–4 h at 20–25 °C. The crude peptide is precipitated from cold diethyl ether, dissolved in water/acetonitrile, and purified by preparative reverse-phase HPLC on C18 columns with 0.1% TFA or 0.1% acetic acid mobile phases. Final materials are lyophilized to moisture ≤ 5% w/w and specified at ≥ 95% HPLC purity, with total Fmoc-related impurities controlled below 0.1% because residual dibenzofulvene can impart an ultraviolet-absorbing contaminant and an off-specification colour reading. The typical scale of these operations ranges from 5 kg to 500 kg of crude peptide per year per line, with solvent consumption dominated by DMF and piperidine. At ton scale, piperidine-containing deprotection waste is segregated and neutralized to pH 6–8 with hydrochloric acid prior to biological treatment; the operation is covered by the facility’s ISO 22716 cosmetic GMP and Regulation (EC) No 1223/2009 obligations for raw material safety. Endotoxin and microbial limits for cosmetic peptide powders follow a risk-based specification of <0.5 EU/mg and total aerobic count <100 CFU/g. The use of Fmoc chemistry rather than Boc chemistry eliminates the need for anhydrous hydrogen fluoride cleavage in final deprotection, reducing the facility safety classification; however, the Fmoc route introduces piperidine handling, which must be engineered with sealed transfer lines and carbon-filtered vent treatment. Published data for exact residual piperidine levels in cosmetic peptide lots is limited because the analytical release focus is typically HPLC purity, TFA content, and water. Process weaknesses are observed most frequently in short peptides rich in glycine and histidine, where coupling efficiency drops unless the resin is pre-swollen in DMF for 30–60 min and the first amino acid loading is limited to 0.4 mmol/g or less. The resulting raw material is then lyophilized into an acetate salt by repeated dissolution in 0.1 mol/L acetic acid and freeze-drying, which reduces residual TFA to <0.1% w/w for cosmetic formulation compatibility.
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Fluorenyl methoxycarbonyl succinimide, systematically named N-(9-fluorenylmethoxycarbonyloxy)succinimide and abbreviated Fmoc-OSu, is a crystalline carbonate reagent used to install the 9-fluorenylmethoxycarbonyl protecting group on primary and secondary amines. Catalog designations include Fmoc succinimidyl carbonate, 9-fluorenylmethyl N-succinimidyl carbonate, and Fmoc-ONSu; all refer to CAS 82911-69-1. The molecular formula is C19H15NO5, and the molecular weight is 337.33 g/mol. Commercial peptide synthesis grade material is commonly supplied as a white to off-white crystalline powder with HPLC purity ≥98.0% at 254 nm, a melting range of 151–155°C by USP <741>, and moisture content ≤0.50% by Karl Fischer titration according to ASTM E203-16. Storage is specified at 2–8°C in a desiccated container under inert gas. The solid should be warmed to room temperature under dry nitrogen before opening to prevent condensation on the powder surface.
The product is commonly supplied in amber glass jars or fluoropolymer-lined containers with a nitrogen blanket. Typical package sizes range from 25 g for laboratory research to 1 kg for pilot-scale campaigns; bulk quantities are shipped in double polyethylene bags inside fiber drums with desiccant. Each package label includes the CAS registry number 82911-69-1, lot number, retest date, and storage statement. The retest date is commonly assigned at 24 months from manufacture when stored at 2–8°C in unopened containers. Opened containers are retested for water content and HPLC purity before use in validated syntheses.
In peptide synthesis, the reagent is used primarily to prepare Fmoc-protected amino acid building blocks from free amino acids under mildly basic aqueous-organic conditions. Published protocols commonly report dissolving the amino acid in dioxane–water or acetonitrile–water containing sodium carbonate or sodium bicarbonate, then adding 1.1–1.5 equivalents of Fmoc-OSu at 0–25°C. Reaction times of 4–24 h are used depending on the steric hindrance of the amino acid side chain. The N-hydroxysuccinimide leaving group is released as a neutral water-soluble species, so the workup consists of pH adjustment and extraction rather than neutralization of hydrogen chloride. The reagent is selective for amino groups over aliphatic hydroxyls under these buffered conditions. It is not compatible with primary or secondary amines used as additives because those amines compete for acylation and generate unwanted Fmoc adducts.
For amino acids with hindered side chains, such as valine or α-aminoisobutyric acid, published protocols report reaction times at the upper end of the range and occasionally require a second addition of 0.5 equivalent Fmoc-OSu after 12 h to reach complete conversion. Reaction progress is monitored by thin-layer chromatography or HPLC; the disappearance of the ninhydrin-positive free amine spot or peak is used as the endpoint. If free amino acid remains after 24 h, the mixture is not held longer because hydrolysis of the reagent competes with acylation. Instead, the reaction is worked up, and the crude product is purified or the unreacted amino acid is recovered from the aqueous phase.
Hydrolysis is the principal competing pathway. In aqueous dioxane or acetonitrile buffered with sodium bicarbonate, the rate of Fmoc-OSu aminolysis must be maintained above the rate of carbonate hydrolysis. The pH is therefore controlled between 8.0 and 9.5. Above pH 10, hydroxide-mediated hydrolysis accelerates and liberates 9-fluorenylmethanol. Under the correct pH window, the free amino group of the amino acid reacts with the carbonate carbonyl to form a stable Fmoc carbamate. The leaving group, N-hydroxysuccinimide, exhibits low absorbance at 254 nm, which simplifies HPLC monitoring but does not eliminate the need to remove residual N-hydroxysuccinimide by aqueous extraction. Process deviations reported on pilot-scale synthesis include emulsion formation during ethyl acetate workup when the aqueous phase is not saturated with sodium chloride. This is resolved by adding 5–10 wt% sodium chloride to the aqueous phase before extraction. Mechanical stirring in glass-lined reactors at 50–100 L scale is commonly specified; high-shear homogenization is not required because the reagent dissolves rapidly in the organic phase.
Because the reagent is moisture-sensitive, the largest batch-to-batch variation is water content. Incoming lots exposed briefly to ambient air can show a decrease in HPLC purity of 0.3–0.8 area% and a corresponding increase in Fmoc-OH content. On a 100 L glass-lined reactor, a charge of Fmoc-OSu that is not equilibrated to room temperature before opening can condense atmospheric moisture on the powder surface. The resulting surface hydrolysis is observed as an off-spec melting point or as a broad melting endotherm by differential scanning calorimetry. For this reason, production sampling is performed under dry nitrogen, and the container is resealed immediately after dispensing.
The following parameters are typically listed on a certificate of analysis for Fmoc-OSu intended for solid-phase peptide synthesis. Specifications vary by supplier and application; the values below represent common commercial acceptance limits rather than a universal standard.
| Parameter | Method | Typical acceptance limit |
|---|---|---|
| Purity | HPLC-UV, 254 nm, area normalization | ≥98.0% |
| Melting range | USP <741> | 151–155°C |
| Water content | Karl Fischer coulometry, ASTM E203-16 | ≤0.50% |
| Appearance | Visual inspection | White to off-white crystalline powder |
| Residue on ignition | USP <281> | ≤0.10% |
| Solubility | 100 mg/mL in DMF at 25°C | Clear, pale yellow solution |
For HPLC purity, reversed-phase C18 columns with acetonitrile–water gradients containing 0.1% trifluoroacetic acid are used. The Fmoc chromophore absorbs strongly at 254 nm, which permits low-level detection of related impurities; however, non-Fmoc impurities without a chromophore may be under-represented by area normalization. Therefore, moisture and residue-on-ignition data are used to confirm that a high HPLC purity value is not masking inorganic or volatile non-chromophoric material. Melting range is determined at a heating rate of 1.0°C/min in a capillary apparatus or by differential scanning calorimetry. A broad melting range greater than 3°C is treated as a signal of residual solvents, moisture, or N-hydroxysuccinimide contamination.
Fmoc-OSu dissolves freely in DMF, DMSO, and dichloromethane; solubility in ethyl acetate and tetrahydrofuran is sufficient for extraction and washing but not always for reaction-scale dissolution. Aqueous solubility is low, and the solid hydrolyzes in water over time. Anhydrous DMF is preferred for reactions with moisture-sensitive substrates. Pre-drying DMF over 3A molecular sieves or using fresh anhydrous DMF stored under argon is recommended when the reagent is used for N-terminal protection of amino esters or amino alcohols. Dichloromethane solutions can be dried over sodium sulfate after aqueous workup without significant product loss, but prolonged storage of Fmoc-OSu in dichloromethane is not recommended because trace moisture in the solvent can cause slow carbamate cleavage.
Fmoc-OSu differs from 9-fluorenylmethyl chloroformate (Fmoc-Cl, CAS 28920-43-6) in physical form, by-product identity, and handling requirements. Fmoc-Cl is a low-melting solid at ambient temperature and generates hydrogen chloride during acylation. Fmoc-OSu is a crystalline solid with a melting range of 151–155°C and releases N-hydroxysuccinimide. The absence of hydrogen chloride eliminates the need for tertiary amine scavengers and reduces acid-catalyzed side reactions during Fmoc protection of acid-sensitive substrates. Fmoc-OSu is less reactive than Fmoc-Cl, so reaction times are longer but racemization is lower under equivalent aqueous-base conditions. For process-scale work, Fmoc-OSu is preferred where operator exposure to lachrymatory acyl chlorides is restricted; however, its higher molecular weight means that 1.1–1.5 equivalents on a molar basis corresponds to a larger mass charge per batch than Fmoc-Cl.
| Property | Fmoc-OSu | Fmoc-Cl |
|---|---|---|
| CAS registry number | 82911-69-1 | 28920-43-6 |
| Molecular weight | 337.33 g/mol | 258.70 g/mol |
| Physical form | White to off-white crystalline solid | White to pale yellow low-melting solid |
| Byproduct | N-hydroxysuccinimide | Hydrogen chloride |
| Moisture sensitivity | Hydrolyzes in aqueous base; store at 2–8°C | Hydrolyzes rapidly; lachrymatory |
| Typical purity | ≥98.0% | ≥98.0% |
| Process advantage | No HCl release, lower racemization | Faster acylation |
Compared with Fmoc chloride, Fmoc-OSu produces a cleaner reaction profile in aqueous carbonate systems. The N-hydroxysuccinimide by-product is water-soluble and can be removed by bicarbonate washes; hydrogen chloride requires neutralization and can protonate unreacted amino acid, altering the pH and slowing the reaction. In reactions with acid-sensitive substrates such as amino acid tert-butyl esters, the hydrogen chloride generated by Fmoc-Cl can cleave the tert-butyl ester; Fmoc-OSu avoids this problem because no strong acid is released. Published comparative studies on Fmoc protection of amino acids report lower racemization with Fmoc-OSu than with Fmoc-Cl under aqueous carbonate conditions; however, specific enantiomeric excess values depend on side-chain structure and published data for this specific configuration is limited for uncommon amino acids.
Fmoc-OSu is not interchangeable with Boc-OSu in orthogonal protection. Boc-OSu installs the tert-butoxycarbonyl group, which is removed under acidic conditions such as trifluoroacetic acid; Fmoc is removed under mild base. This difference is used in peptide synthesis to achieve selective deprotection. An Fmoc-protected amine can be carried through a Boc removal step using trifluoroacetic acid when the Fmoc group remains stable to acid. Conversely, a Boc-protected amine is not compatible with the piperidine conditions used for Fmoc removal.
Fmoc-OSu is also distinguished from Fmoc-pentafluorophenyl carbonate and other active carbonates by the N-hydroxysuccinimide leaving group. The pentafluorophenyl analog is more reactive in some organic media, but its by-product is a weakly acidic phenol that can complicate aqueous workup and may require additional extraction under highly basic conditions. The N-hydroxysuccinimide by-product from Fmoc-OSu is neutral and water-soluble, which simplifies purification. However, the pentafluorophenyl analog may be preferred when rapid reaction with hindered secondary amines is required; published data for this specific configuration is limited, and solvent compatibility must be evaluated case by case.
When anhydrous processing is not maintained, Fmoc-OSu undergoes hydrolysis to 9-fluorenylmethanol and N-hydroxysuccinimide. The reaction becomes measurable in DMF containing greater than 0.1% water after 24 h at 25°C. Pre-drying DMF over 3A molecular sieves or using fresh anhydrous DMF stored under argon is therefore required when the reagent is used for N-terminal protection of amino esters or amino alcohols. The reagent should not be combined with nucleophilic catalysts such as 4-dimethylaminopyridine in aqueous systems because base catalysis accelerates carbonate hydrolysis. Storage is at 2–8°C in a desiccator with silica gel or molecular sieves. Repeated freeze-thaw cycling is not recommended because condensation during temperature equilibration can raise water content. Before opening, the container is warmed to room temperature under dry nitrogen. If water content exceeds the specification, the material is not suitable for reactions that require stoichiometric control of the Fmoc group.
Fmoc-OSu is used to prepare Fmoc-protected amino acids, amino alcohols, and hydrazines for solid-phase synthesis. The resulting Fmoc carbamate is stable to acidic side-chain deprotection conditions, but it is removed by 20% piperidine in DMF in 10–20 min at ambient temperature. In process-scale solid-phase synthesis, complete Fmoc deprotection is monitored by the UV absorbance of the dibenzofulvene–piperidine adduct at 301 nm; this in-process control is typical in automated peptide synthesizers equipped with a UV detector. The first deprotection cycle often shows a higher absorbance because residual Fmoc-protected amino acid from incomplete coupling is also cleaved. Automated peptide synthesizers use this signal to stop the deprotection step when the absorbance falls below a set threshold. For manual synthesis, a negative Kaiser test after coupling and a positive UV signal after deprotection are used as in-process controls. Fmoc-OSu is therefore selected when temporary Nα protection must be removed on-resin without releasing side-chain protecting groups. The operational boundary is defined by moisture ingress, pH above 10, and the presence of free amines in storage or reaction mixtures. Published data for this specific configuration is limited for non-peptide substrates, so compatibility tests are performed before scale-up.
